The present invention relates to a process for producing N-methyl-L-alanine derivatives and to novel process intermediates.
Processes for the preparation of N-methyl-L-alanine derivatives from dithioalkanoic acid intermediates and N-methyl-L-alanine have been reported in U.S. Pat. No. 5,208,020. The compounds are useful for linking cytotoxic maytansinoids to cell-binding agents such as antibodies. Antibody/maytansinoid complexes are useful as tumor-activated pro-drugs.
For example, the multi-step synthesis of N-methyl-N-(3-methyldithio-propanoyl)-L-alanine has been disclosed. First, 3-methyldithiopropanoic acid was prepared by adding methyl methanethiosulfonate in ethanol to a solution of 3-mercaptopropanoic acid in water. After extraction, washing and concentration, the 3-methyldithiopropanoic acid was isolated by distillation. Isobutylchloroformate and triethylamine were added to the 3-methyldithiopropanoic acid in THF to form the corresponding mixed anhydride as intermediate. Subsequently, a mixture of N-methyl-L-alanine and triethylamine in water was added. After extraction, oncentration and chromatography, a 34% yield of N-methyl-N-(3-methyldithio-propanoyl)-L-alanine was obtained.
A major shortcoming of the prior art processes is the necessity of two chromatography steps to remove side products from the desired reaction products. Further, the use of isobutylchloroformate in the reaction scheme allows for racemization which leads to a final product containing the undesired D-enantiomer. Thus, there is a need in the art for improved methods to prepare N-methyl-L-alanine derivatives with an optical purity  greater than 95% enantiomeric excess (ee) where the intermediates are more stable, resulting in less undesired side products.
One aspect of the invention is processes for the preparation of N-methyl-L-alanine derivatives.
Another aspect of the invention is novel intermediates useful for the preparation of N-methyl-L-alanine derivatives.
Another aspect of the invention is processes for the preparation of the novel intermediates of the invention.
Yet another aspect of the invention is cell-binding agent/maytansinoid complexes prepared from the N-methyl-L-alanine derivatives or novel intermediates produced by the processes of the invention.
All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as though fully set forth.
By the term xe2x80x9ccoupling reagentxe2x80x9d as used herein and in the claims is meant a compound or compounds which are capable of forming an activated derivative of methyldithiopropionic acid or its homologues which is stable to hydrolysis under the employed reaction conditions and yields a compound of Formula I with an optical purity  greater than 95% ee when reacted with N-methyl-L-alanine.
The present invention provides a process for the preparation of compounds of Formula I 
M is a direct bond, CH2, 
a is 0 or an integer of 1 to 9 when M is a direct bond provided that when M is one or more carbon atoms a is 0 or an integer of 1 to 8;
d is an integer of 3 to 8; and
Z is H or SR2, where R2 is linear alkyl, branched alkyl, cyclic alkyl, aryl, substituted aryl or heterocyclic.
The present invention also provides novel intermediates that are useful in the process of the invention.
The present invention also provides antibody/maytansinoid complexes produced by the process of the invention.
The process of the invention comprises the steps of reacting a salt of a mercaptoalkanoic acid of Formula II 
where L is as defined above;
and thiolsulfonates of Formula III
QSO2SQxe2x80x83xe2x80x83(III) 
where Q is H, linear alkyl, branched alkyl, cyclic alkyl, aryl, substituted aryl or heterocyclic,
in a disulfide formation reaction where a salt of a compound of Formula II in water is added to a compound of Formula III in a water-immiscible polar organic solvent to form intermediates of Formula IV 
where L and Q are as defined above; and
reacting a compound of Formula IV in an esterification reaction with a coupling reagent such as that of Formula V 
where
R3 is H or substituted uronium salt of the formula 
where Xxe2x88x92 is PF6xe2x88x92 or BF4xe2x88x92, R7 and R8 are independently linear alkyl, branched alkyl, cycloalkyl or (CH2)e where e is an integer of 3 to 8 with the proviso that when R7 is (CH2)e, R8 is a direct bond,
or a substituted phosphonium salt of the formula 
where Xxe2x88x92 is PF6xe2x88x92 or BF4xe2x88x92, R9 and R10 are independently linear alkyl, branched alkyl, cycloalkyl or heterocyclic; and
R4 and R5 are independently H, a double bond, linear alkyl, branched alkyl, cyclic alkyl, aryl, substituted aryl or heterocyclic.
If R3 is H, the reaction is carried out in the presence of a carbodiimide reagent of Formula VI 
where
R11 and R12 are independently linear alkyl, branched alkyl, cyclic alkyl, aryl, substituted aryl or heterocyclic. Preferably, the carbodiimide reagent of Formula VI is 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDCI.HCl) where R11 is ethyl and R12 is dimethylaminopropyl or 1,3-dicyclohexylcarbodiimide (DCC) where R11 and R12 are cyclohexyl. Particularly preferred is EDCI.HCl.
Alternatively, if R3 is a uronium or phosphonium salt, the reaction is carried out in the presence of a base, preferably triethylamine or diisopropylethylamine.
These reactions form compounds of Formula VII 
where R4, R5, L and Q are as defined above.
Compounds of Formula VII are activated esters and are novel intermediates of the process of the invention and are stable to hydrolysis under the employed reaction conditions and allow racemization-free coupling with N-methyl-alanine. Preferably, the compounds are crystalline.
Compounds of Formula VII are subsequently reacted with N-methyl-L-alanine (Formula VIII) 
to form a compound of Formula I.
Further, the present invention also includes a process for the preparation of compounds of Formula I comprising reacting a compound of Formula VII with N-methyl-L-alanine to form a compound of Formula I and compounds of Formula I prepared by the process.
The present invention also includes a process for the preparation of a compound of Formula VII 
where
R4 and R5 are independently H, a double bond, linear alkyl, branched alkyl, cyclic alkyl, aryl, substituted aryl or heterocyclic, 
M is a direct bond, CH2, 
a is 0 or an integer of 1 to 9 when M is a direct bond provided that when M is one or more carbon atoms a is 0 or an integer of 1 to 8; and
d is an integer of 3 to 8; and
Q is H, linear alkyl, branched alkyl, cyclic alkyl, simple or substituted aryl or hetero cyclic which comprises the steps of:
(1) reacting a salt of a mercaptoalkanoic acid of Formula II 
where L is as defined above;
and thiolsulfonates of Formula III
QSO2SQxe2x80x83xe2x80x83(III) 
where Q is as defined above,
in a disulfide formation reaction where a salt of a compound of Formula II in water is added to a compound of Formula III in a water-immiscible polar organic solvent to form intermediates of Formula IV 
where L and Q are as defined above; and
(2) reacting a compound of Formula IV in an esterification reaction with a coupling reagent of Formula V 
where
R3 is H or substituted uronium salt of the formula 
where Xxe2x88x92 is PF6xe2x88x92 or BF4xe2x88x92, R7 and R8 are independently alkyl, cycloalkyl or (CH2)e, where e is an integer of 3 to 8 with the proviso that when R7 is (CH2)e, R8 is a direct bond,
or a substituted phosphonium salt of the formula 
where Xxe2x88x92 is PF6xe2x88x92 or BF4xe2x88x92, R9 and R10 are independently linear alkyl, branched alkyl, cycloalkyl or heterocyclic; and
R4 and R5 are as defined above,
provided that when R3 is H, the reaction is carried out in the presence of a carbodiimide reagent of Formula VI
R11xe2x80x94Nxe2x95x90Cxe2x95x90Nxe2x80x94R12xe2x80x83xe2x80x83(VI) 
where
R11 and R12 are independently linear alkyl, branched alkyl, cyclic alkyl, aryl, substituted aryl or heterocyclic, and
further provided that if R3 is a uronium or phosphonium salt, the reaction is carried out in the presence of a base,
to form compounds of Formula VII.
Examples of linear alkyls include methyl, ethyl, propyl, butyl, pentyl and hexyl. Examples of branched alkyls include isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl and 1-ethyl-propyl. Examples of cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
Examples of aryls include phenyl and naphthyl. Examples of substituted aryls include aryls substituted with alkyl, halogen (such as chlorine, bromine and iodine), nitro, amino, sulfonic acid, carboxylic acid, hydroxy and alkoxy. 
Heteroatoms are selected from O, N and S. Examples of heterocyclics include pyrrollyl, pyridyl, furyl and thiophene.
Compounds of Formula II and their salts can be prepared by known methods from readily available starting materials such as described in U.S. Pat. No. 5,208,020.
Compounds of Formula III such as methyl methanethiolsulfonate and S-phenyl benzenethiolsulfonate can be purchased from chemical supply houses such as Aldrich Chemical Co. (Milwaukee, Wis.) or prepared from readily available starting materials by known disulfide oxidation methods such as described in Xia et al., Synth. Commun. 27, 1301-1308 (1997) and Pinnick et al., J. Org. Chem. 45, 930-932 (1980) provided that Q does not include any moieties that render inoperative the process of the invention.
Exemplary compounds of Formula V are:
N-hydroxysuccinimide (Va);
O-(N-succinimidyl)-1,1,3,3-bis(tetramethylene)uronium tetrafluoroborate or its corresponding hexafluorophosphate (Vb);
O-(N-succinimidyl)-1,1,3,3-tetramethyluronium-tetrafluoroborate or its corresponding hexafluorophosphate (Vc);
N-hydroxy-5-norbornene-2,3-dicarboxylic acid imide (Vd);
O-(5-Norbornene-2,3-dicarboximido)-1,1,3,3-tetrametyluronium tetrafluoroborate (Ve);
O-(5-Norbornene-2,3-dicarboximido)-1,1,3,3-tetrametyluronium hexafluorophosphate derivative (Vf);
N-hydroxyphthalimide (Vg);
1,1,3,3-tetramethyluronium tetrafluoroborate N-hydroxyphthalimide (Vh);
1,1,3,3-tetramethyluronium hexafluorophosphate N-hydroxyphthalimide (Vi);
N,N,Nxe2x80x2,Nxe2x80x2-bis(pentamethylene)uronium tetrafluoroborate N-hydroxyphthalimide (Vj);
N,N,Nxe2x80x2,Nxe2x80x2-bis(pentamethylene)uronium hexafluorophosphate N-hydroxyphthalimide (Vk);
N,N,Nxe2x80x2,Nxe2x80x2-bis(tetramethylene)uronium tetrafluoroborate N-hydroxyphthalimide (Vl);
N,N,Nxe2x80x2,Nxe2x80x2-bis(tetramethylene)uronium hexafluorophosphate N-hydroxyphthalimide (Vm);
N,N,Nxe2x80x2,Nxe2x80x2-tetramethyluronium tetrafluoroborate (Vn);
N,N,Nxe2x80x2,Nxe2x80x2-tetramethyluronium hexafluorophosphate (Vo);
tris(dimethylamino)phosphonium tetrafluoroborate (Vp);
tris(dimethylamino)phosphonium hexafluorophosphate (Vq);
tripyrrolidinophosphonium tetrafluoroborate (Vr);
tripyrrolidinophosphonium hexafluorophosphate (Vs);
1,3-dimethylimidazolidinium tetrafluoroborate (Vt); and
1,3-dimethylimidazolidinium hexafluorophosphate (Vu).
Compounds of Formula V can be purchased from chemical supply houses such as Fluka Chemical Corp. (Milwaukee, Wis.) or prepared by known methods from readily available starting materials. For example, compounds of Formula V can be prepared by the method of Knorr et al. in Tetrahedron Lett. 30, 1927-1930 (1989).
Compounds of Formula VI can be purchased from chemical supply houses such as Aldrich Chemical Co. (St. Louis, Mo.) or prepared by known methods from readily available starting materials.
The compound of Formula VIII (N-methyl-L-alanine) can be purchased from BACHEM Bioscience Inc. (King of Prussia, Pa.).
The reaction to convert compounds of Formula II to compounds of Formula IV is carried out by adding an aqueous solution of a salt of a compound of Formula II to a compound of Formula III in a water-immiscible polar organic solvent, preferably at a temperature range of less than xe2x88x925xc2x0 C. Most preferably, an aqueous solution of the sodium salt of a compound of Formula II is added slowly to a solution of the compound of Formula III in tetrahydrofuran at a temperature range of about xe2x88x9215xc2x0 C. to about xe2x88x925xc2x0 C. A particularly preferred temperature range is about xe2x88x9210xc2x0 C. to about xe2x88x925xc2x0 C.
Preferably, the reaction to convert compounds of Formula IV to the activated esters of Formula VII, when R3 of Formula V is H, is carried out in the presence of a carbodiimide reagent at a temperature range of about 0xc2x0 C. to about 20xc2x0 C., preferably about 0xc2x0 C. to about 12xc2x0 C., in a solvent such as methylene chloride. When R3 of Formula V is a uronium or phosphonium salt, the reaction is carried out in the presence of a base, preferably triethylamine or diisopropylethylamine.
Preferably, the reaction to convert compounds of Formula VII to compounds of Formula I is carried out at ambient temperature in a polar solvent such as aqueous ethanol/triethylamine.
The reagents, conditions and intermediates used in the present invention provide for high yields of the desired compounds of Formula I making the process appropriate for industrial scale utilization. Further, the process of the invention has other advantages over the processes disclosed in U.S. Pat. No. 5,208,020. Specifically, the claimed process converts compounds of Formula II to compounds of Formula IV without the formation of undesired side products of symmetric disulfides of thioproprionic acid or its homologues. Accordingly, no isolation step for compounds of Formula IV is necessary and a one-pot process can be used to convert compounds of Formula II to the novel intermediates of Formula VII, which can be isolated. Alternatively, the Formula VII intermediate is not isolated and a one-pot process can be used to convert compounds of Formula II to compounds of Formula I.
Further, the claimed process affords the activated esters of Formula VII which are crystalline and stable to hydrolysis. Accordingly, the compounds of Formula I produced by the reaction of N-methyl-L-alanine with the compounds of Formula VII are substantially optically pure L-isomer.
Most preferably, the process of the present invention is used for preparing a compound of Formula Ia which is N-methyl-N-(3-methyldithiopropanoyl)-L-alanine 
The preferred process is shown schematically below. 
The process of the invention can be used to make cell-binding agent/maytansanoid complexes which are useful as tumor-activated pro-drugs. Compounds of Formula VII can be converted into a cell-binding agent/maytansinoid complex. Further, compounds of Formula I produced by the process of the invention are used as described in U.S. Pat. No. 5,208,020 to produce N-methyl-L-alanine containing maytansanoid derivatives. These derivatives are then conjugated to cell-binding agents, preferably antibodies, via various linkers. Preferably, the linkage is a disulfide link.
An exemplary cell-binding agent/maytansinoid complex can be prepared by a process comprising the following steps:
(1) esterifying maytansinol with a compound of Formula I prepared by the process of the invention to form a disulfide-containing maytansinoid ester;
(2) reducing the disulfide-containing maytansinoid ester prepared by step (1) to a thiol-containing maytansinoid;
(3) introducing dithiopyridyl groups into a cell-binding agent; and
(4) linking the thiol-containing maytansinoid produced by step (2) to the dithiopyridyl cell-binding agent of step (3) by a disulfide link.
The present invention will now be described with reference to the following specific, non-limiting example.